Synergistic Nanomedicine Delivering Topoisomerase I Toxin (SN-38) and Inhibitors of Polynucleotide Kinase 3'-Phosphatase (PNKP) for Enhanced Treatment of Colorectal Cancer.

Inhibitors of a DNA repair enzyme known as polynucleotide kinase 3'-phosphatase (PNKP) are expected to show synergistic cytotoxicity in combination with topoisomerase I (TOP1) inhibitors in cancer. In this study, the synergistic cytotoxicity of a novel inhibitor of PNKP, i.e., A83B4C63, with a potent TOP1 inhibitor, i.e., SN-38, against colorectal cancer cells was investigated. Polymeric micelles (PMs) for preferred tumor delivery of A83B4C63, developed through physical encapsulation of this compound in methoxy poly(ethylene oxide)-poly(α-benzyl carboxylate-ε-caprolactone) (mPEO-b-PBCL) micelles, were combined with SN-38 in free or PM form. The PM form of SN-38 was prepared through chemical conjugation of SN-38 to the functional end group of mPEO-b-PBCL and further assembly of mPEO-b-PBCL-SN-38 in water. Moreover, mixed micelles composed of mPEO-b-PBCL and mPEO-b-PBCL-SN-38 were used to co-load A83B4C63 and SN-38 in the same nanoformulation. The loading content (% w/w) of the SN-38 and A83B4C63 to mPEO-b-PBCL in the co-loaded formulation was 7.91 ± 0.66 and 16.13 ± 0.11% (w/w), respectively, compared to 15.67 ± 0.34 (% w/w) and 23.06 ± 0.63 (% w/w) for mPEO-b-PBCL micelles loading individual drugs. Notably, the average diameter of PMs co-encapsulating both SN-38 and A83B4C63 was larger than that of PMs encapsulating either of these compounds alone but still lower than 60 nm. The release of A83B4C63 from PMs co-encapsulating both drugs was 76.36 ± 1.41% within 24 h, which was significantly higher than that of A83B4C63-encapsulated micelles (42.70 ± 0.72%). In contrast, the release of SN-38 from PMs co-encapsulating both drugs was 44.15 ± 2.61% at 24 h, which was significantly lower than that of SN-38-conjugated PMs (74.16 ± 3.65%). Cytotoxicity evaluations by the MTS assay as analyzed by the Combenefit software suggested a clear synergy between PM/A83B4C63 (at a concentration range of 10-40 µM) and free SN-38 (at a concentration range of 0.001-1 µM). The synergistic cytotoxic concentration range for SN-38 was narrowed down to 0.1-1 or 0.01-1 µM when combined with PM/A83B4C63 at 10 or 20-40 µM, respectively. In general, PMs co-encapsulating A83B4C63 and SN-38 at drug concentrations within the synergistic range (10 µM for A83B4C63 and 0.05-1 µM for SN-38) showed slightly less enhancement of SN-38 anticancer activity than a combination of individual micelles, i.e., A83B4C63 PMs + SN-38 PMs at the same molar concentrations. This was attributed to the slower release of SN-38 from the SN-38 and A83B4C63 co-encapsulated PMs compared to PMs only encapsulating SN-38. Cotreatment of cells with TOP1 inhibitors and A83B4C63 formulation enhanced the expression level of γ-HA2X, cleaved PARP, caspase-3, and caspase-7 in most cases. This trend was more consistent and notable for PMs co-encapsulating both A83B4C63 and SN-38. The overall result from the study shows a synergy between PMs of SN-38 and A83B4C63 as a mixture of two PMs for individual drugs or PMs co-encapsulating both drugs.

Nano-delivery of Silibinin Potentiate the Induction of Immunogenic Cell Death (ICD) in Melanoma Cells.

BACKGROUND: Induction of immunogenic cell death (ICD) in tumors can enhance antitumor immunity and modulate immunosuppression in the tumor microenvironment (TME). OBJECTIVE: In the current study, we investigated the effect of silibinin, a natural compound with anticancer activity, and its polymer-based nanoformulations on the induction of apoptosis and ICD in cancer cells. METHODS: Free and nanoparticulate silibinin were evaluated for their growth-inhibitory effects using an MTT assay. Annexin V/PI staining was used to analyze apoptosis. Calreticulin (CRT) expression was measured by flow cytometry. Western blotting was conducted to examine the levels of elf2α, which plays a role in the ICD pathway. The HSP90 and ATP levels were determined using specific detection kits. RESULTS: Compared to the free drug, silibinin-loaded nanocarriers significantly increased the induction of apoptosis and ICD in B16F10 cells. ICD induction was characterized by significantly increased levels of ICD biomarkers, including CRT, HSP90, and ATP. We also observed an increased expression of p-elf-2α/ elf-2α in B16F10 cells treated with silibinin-loaded micelles compared to cells that received free silibinin. CONCLUSION: Our findings showed that the encapsulation of silibinin in polymeric nanocarriers can potentiate the effects of this drug on the induction of apoptosis and ICD in B16F10 melanoma cells.

Peptide-Drug Conjugate Targeting Keratin 1 Inhibits Triple-Negative Breast Cancer in Mice.

Selective delivery of chemotherapy to the tumor site while sparing healthy cells and tissues is an attractive approach for cancer treatment. Carriers such as peptides can facilitate selective tumor targeting and payload delivery. Peptides with specific affinity for the overexpressed cell-surface receptors in cancer cells are conjugated to chemotherapy to afford peptide-drug conjugates (PDCs) that show selective uptake by cancer cells. Using a 10-mer linear peptide (WxEAAYQrFL) called 18-4 that targets and binds breast cancer cells, we designed a peptide 18-4-doxorubicin (Dox) conjugate with high specific toxicity toward triple-negative breast cancer (TNBC) MDA-MB-231 cells and 30-fold lower toxicity to normal breast MCF10A epithelial cells. Here, we elucidate the in vivo activity of this potent and tumor-selective peptide 18-4-Dox conjugate in mice bearing orthotopic MDA-MB-231 tumors. Mice treated with four weekly injections of the conjugate showed significantly lower tumor volumes compared to mice treated with free Dox at an equivalent Dox dose. Immunohistochemical (IHC) analysis of mice tissues revealed that treatment with a low dose of PDC (2.5 mg/kg of Dox equiv) reduced the expression of proliferation markers (PCNA and Ki-67) and increased apoptosis (evidenced by increased caspase-3 expression). At the same dose of free Dox (2.5 mg/kg), the expression of these markers was similar to that of saline treatment. Accordingly, significantly more Dox accumulated in tumors of conjugate-treated mice (7-fold) compared to the Dox-treated mice, while lower levels of Dox were observed in the liver, heart, and lungs of peptide-Dox conjugate-treated mice (up to 3-fold less) than Dox-treated mice. The IHC analysis of keratin 1 (K1), the receptor for peptide 18-4, revealed K1 upregulation in tumors and low levels in normal mammary fat pad and liver tissues from mice, suggesting preferential uptake of PDCs by TNBC to be K1 receptor-mediated. Taken together, our data support the use of a PDC approach to deliver chemotherapy selectively to the TNBC to inhibit tumor growth.

Repurposing Antimalarial Pyronaridine as a DNA Repair Inhibitor to Exploit the Full Potential of Gold-Nanoparticle-Mediated Radiation Response.

Radiation therapy (RT) is frequently used to locally treat tumors. One of the major issues in RT is normal tissue toxicity; thus, it is necessary to limit dose escalation for enhanced local control in patients that have locally advanced tumors. Integrating radiosensitizing agents such as gold nanoparticles (GNPs) into RT has been shown to greatly increase the cure rate of solid tumors. The objective of this study was to explore the repurposing of an antimalarial drug, pyronaridine (PYD), as a DNA repair inhibitor to further enhance RT/GNP-induced DNA damage in cancerous cell lines. We were able to achieve inhibitory effects of DNA repair due to PYD at 500 nM concentration. Our results show a significant enhancement in DNA double-strand breaks of 42% in HeLa cells treated with PYD/GNP/RT in comparison to GNP/RT alone when irradiated with a dose of 2 Gy. Furthermore, there was a significant reduction in cellular proliferation for both HeLa and HCT-116 irradiated cells with the combined treatment of PYD/GNP/RT. Therefore, the emergence of promising novel concepts introduced in this study could lay the foundation for the transition of this treatment modality into clinical environments.

An injectable thermosensitive hydrogel/nanomicelles composite for local chemo-immunotherapy in mouse model of melanoma.

Recently, cancer immunotherapy and its combination with chemotherapy has been considered to improve therapeutic efficacy with lower systemic toxicity. Here, we prepared a thermosensitive hydrogel based hyaluronic acid (HA) encapsulated with macrophage colony-stimulating factor (GM-CSF) and paclitaxel (PTX) for chemoimmunotherapy of cancer. For this purpose, the micelles were prepared with the mixture of pluronic F127 (PF127) and tocopheryl polyethylene glycol (TPGS) and loaded with PTX. In the following step, thermosensitive hydrogel using PF127 and HA was prepared and co-encapsulated with the micelles and GM-CSF. Rheological performance, friability, release patterns for PTX and GM-CSF, and stability of GM-CSF in the hydrogel were evaluated in details. In-vitro and in vivo immunologic activities of GM-CSF in the hydrogel were also evaluated via numbering macrophages and recruited DCs in transwells and after subcutaneous injection of the GM-CSF-loaded hydrogel. Finally, mouse model of subcutaneous melanoma was induced in female C57 mice using B16 F10 cell line and the effect of optimized formulation was evaluated based on tumor volume and histological analysis. The hydrogel could maintain the biological activity of the incorporated drugs and exhibited a more prolonged release for PTX compared to GM-CSF. GM-CSF-releasing HA/PF127 hydrogel successfully recruited macrophages in vitro. Moreover, the most potent anti-tumor effect was observed following the intra-tumoral injection of the optimized formulation in melanoma bearing mice, compared to immunization by the GM-CSF and PTX alone. The current formulation shows a great promise to conquer resistant malignancies and provides a new approach for co-encapsulating of hydrophobic anticancer drugs and growth factor.

Biodistribution and Activity of EGFR Targeted Polymeric Micelles Delivering a New Inhibitor of DNA Repair to Orthotopic Colorectal Cancer Xenografts with Metastasis.

The disruption of polynucleotide kinase/phosphatase (PNKP) in colorectal cancer (CRC) cells deficient in phosphatase and tensin homolog (PTEN) is expected to lead to the loss of cell viability by a process known as synthetic lethality. In previous studies, we have reported on the encapsulation of a novel inhibitor of PNKP, namely, A83B4C63, in polymeric micelles and its activity in slowing the growth of PTEN-deficient CRC cells as well as subcutaneous xenografts. In this study, to enhance drug delivery and specificity to CRC tumors, the surface of polymeric micelles carrying A83B4C63 was modified with GE11, a peptide targeting epidermal growth factor receptor (EGFR) overexpressed in about 70% of CRC tumors. Using molecular dynamics (MD) simulations, we assessed the binding site and affinity of GE11 for EGFR. The GE11-modified micelles, tagged with a near-infrared fluorophore, showed enhanced internalization by EGFR-overexpressing CRC cells in vitro and a trend toward increased primary tumor homing in an orthotopic CRC xenograft in vivo. In line with these observations, the GE11 modification of polymeric micelles was shown to positively contribute to the improved therapeutic activity of encapsulated A83B4C63 against HCT116-PTEN-/- cells in vitro and that of orthotopic CRC xenograft in vivo. In conclusion, our results provided proof of principle evidence for the potential benefit of EGFR targeted polymeric micellar formulations of A83B4C63 as monotherapeutics for aggressive and metastatic CRC tumors but at the same time highlighted the need for the development of EGFR ligands with improved physiological stability and EGFR binding.

Three-Dimensional Reconstructed Bone Marrow Matrix Culture Improves the Viability of Primary Myeloma Cells In-Vitro via a STAT3-Dependent Mechanism.

Primary myeloma (PM) cells are short-lived in conventional culture, which limited their usefulness as a study model. Here, we evaluated if three-dimensional (3D) culture can significantly prolong the longevity of PM cells in-vitro. We employed a previously established 3D model for culture of bone marrow mononuclear cells isolated from 15 patients. We assessed the proportion of PM cells, viability and proliferation using CD38 staining, trypan blue exclusion assays and carboxy fluorescein succinimidyl ester (CFSE) staining, respectively. We observed significantly more CD38+ viable cells in 3D than in conventional culture (65% vs. 25%, p = 0.006) on day 3. CFSE staining showed no significant difference in cell proliferation between the two culture systems. Moreover, we found that PM cells in 3D culture are more STAT3 active by measure of pSTAT3 staining (66% vs. 10%, p = 0.008). Treatment of IL6, a STAT3 activator significantly increased CD38+ cell viability (41% to 68%, p = 0.021). In comparison, inhibition of STAT3 with Stattic significantly decreased PM cell viability in 3D culture (38% to 17% p = 0.010). Neither IL6 nor Stattic affected the PM cell viability in conventional culture. This study suggests that 3D culture can significantly improve the longevity of PM cells in-vitro, and STAT3 activation can further improve their viability.

Design and evaluation of albumin nanoparticles for the delivery of a novel ß-tubulin polymerization inhibitor.

PURPOSE: The ultimate goal of this study is to develop a novel delivery system for a new potent cytotoxic compound, CCI-001, with anti-b tubulin activity, so that the drug can be effectively administered and at the same time its harmful side effects can be reduced. METHODS: In the current study, CCI-001 was loaded into serum albumin (SA), using a modified desolvation method, generating CCI-001-SA nanoparticles. Both bovine and human SA were used for the encapsulation of this drug candidate. Optimum conditions for drug loading were achieved when already formed and crosslinked albumin nanoparticles were incubated overnight at 37°C with CCI-001 solutions. The CCI-001-loaded albumin nanoparticles were assessed for average particle diameter and polydispersity, zeta potential, drug loading, in vitro release, morphology and cell toxicity against SW620 and HCT116 colorectal cancer cells. RESULTS: The spherical nanoparticles obtained were negatively charged (~ -30 mV) and had an average diameter of ~ 130 nm, with a narrow size distribution. The in vitro release of CCI-001 from the albumin nanoparticles showed a sustained release pattern over 24 hours without any initial burst release, compared to the fast release of the free drug under experimental conditions. No difference between the SA from the two species in terms of CCI-001 loading was observed. However, a significant difference was observed between the release profiles of CCI-001 from drug-loaded HSA and drug-loaded BSA nanoparticles with HSA nanoparticles showing slower drug release (mean release time, MRT, values of 5.14 ± 0.33 h and 6.88 ± 0.15 h for BSA-NPs and HSA-NPs, respectively, P < 0.01). Cellular toxicity studies showed higher cytotoxicity for CCI-001-SA compared to the free drug (IC50s of 0.62 ± 0.31 nM vs 2.06 ± 0.29 nM in SW620 cells and 0.9 ± 0.1 nM vs 4.2 ± 0.2 nM in HCT116 cells, for CCI-001-HSA NPs and free drug, respectively). Therefore, despite the low drug content level in the HSA nanoparticles of CCI-001, the formulation provides relevant concentrations for further in vivo studies in animal models due to high drug potency. CONCLUSIONS: The data support the potential use of albumin as a nanocarrier for CCI-001 in biological systems.

A synthetically lethal nanomedicine delivering novel inhibitors of polynucleotide kinase 3'-phosphatase (PNKP) for targeted therapy of PTEN-deficient colorectal cancer.

Phosphatase and TENsin homolog deleted on chromosome 10 (PTEN) is a major tumor-suppressor protein that is lost in up to 75% of aggressive colorectal cancers (CRC). The co-depletion of PTEN and a DNA repair protein, polynucleotide kinase 3'-phosphatase (PNKP), has been shown to lead to synthetic lethality in several cancer types including CRC. This finding inspired the development of novel PNKP inhibitors as potential new drugs against PTEN-deficient CRC. Here, we report on the in vitro and in vivo evaluation of a nano-encapsulated potent, but poorly water-soluble lead PNKP inhibitor, A83B4C63, as a new targeted therapeutic for PTEN-deficient CRC. Our data confirmed the binding of A83B4C63, as free or nanoparticle (NP) formulation, to intracellular PNKP using the cellular thermal shift assay (CETSA), in vitro and in vivo. Dose escalating toxicity studies in healthy CD-1 mice, based on measurement of animal weight changes and biochemical blood analysis, revealed the safety of both free and nano-encapsulated A83B4C63, at assessed doses of ≤50 mg/kg. Nano-carriers of A83B4C63 effectively inhibited the growth of HCT116/PTEN-/- xenografts in NIH-III nude mice following intravenous (IV) administration, but not that of wild-type HCT116/PTEN+/+ xenografts. This was in contrast to IV administration of A83B4C63 solubilized with the aid of Cremophor EL: Ethanol (CE), which led to similar tumor growth to that of formulation excipients (NP or CE without drug) or 5% dextrose. This observation was attributed to the higher levels of A83B4C63 delivered to tumor tissue by its NP formulation. Our data provide evidence for the success of NPs of A83B4C63, as novel synthetically lethal nano-therapeutics in the treatment of PTEN-deficient CRC. This research also highlights the potential of successful application of nanomedicine in the drug development process.

The Uniqueness of Albumin as a Carrier in Nanodrug Delivery.

Albumin is an appealing carrier in nanomedicine because of its unique features. First, it is the most abundant protein in plasma, endowing high biocompatibility, biodegradability, nonimmunogenicity, and safety for its clinical application. Second, albumin chemical structure and conformation allows interaction with many different drugs, potentially protecting them from elimination and metabolism in vivo, thus improving their pharmacokinetic properties. Finally, albumin can interact with receptors overexpressed in many diseased tissues and cells, providing a unique feature for active targeting of the disease site without the addition of specific ligands to the nanocarrier. For this reason, albumin, characterized by an extended serum half-life of around 19 days, has the potential of promoting half-life extension and targeted delivery of drugs. Therefore, this article focuses on the importance of albumin as a nanodrug delivery carrier for hydrophobic drugs, taking advantage of the passive as well as active targeting potential of this nanocarrier. Particular attention is paid to the breakthrough NAB-Technology, with emphasis on the advantages of Nab-Paclitaxel (Abraxane), compared to the solvent-based formulations of Paclitaxel, i.e., CrEL-paclitaxel (Taxol) in a clinical setting. Finally, the role of albumin in carrying anticancer compounds is depicted, with a particular focus on the albumin-based formulations that are currently undergoing clinical trials. The article sheds light on the power of an endogenous substance, such as albumin, as a drug delivery system, signifies the importance of the drug vehicle in drug performance in the biological systems, and highlights the possible future trends in the use of this drug delivery system.

Pharmacokinetic and Tissue Distribution of Orally Administered Cyclosporine A-Loaded poly(ethylene oxide)-block-Poly(ε-caprolactone) Micelles versus Sandimmune® in Rats.

PURPOSE: We have previously reported on a polymeric micellar formulation of Cyclosporine A (CyA) based on poly(ethylene oxide)-block-poly(ε-caprolactone) (PEO5K-b-PCL13K) capable of changing drug biodistribution and pharmacokinetic profile following intravenous administration. The objective of the present study was to explore the potential of this formulation in changing the tissue distribution and pharmacokinetics of the encapsulated CyA following oral administration making comparisons with Sandimmune®. METHODS: The in vitro CyA release and stability CyA-loaded PEO-b-PCL micelles (CyA-micelles) were evaluated in biorelevant media. The pharmacokinetics and tissue distribution of orally administered CyA-micelles or Sandimmune® and tissue distribution of traceable Cyanine-5.5 (Cy5.5)-conjugated PEO-b-PCL micelles were then investigated in healthy rats. RESULTS: CyA-micelles showed around 60-70% CyA release in simulated intestinal and gastric fluids within 24 h, while Sandimmune® released its entire CyA content in the simulated intestinal fluid. CyA-micelles and Sandimmune® showed similar pharmacokinetics, but different tissue distribution profile in rats. In particular, the calculated AUC for CyA-micelles was higher in liver, comparable in heart, and lower in spleen, lungs, and kidneys when compared to that for Sandimmune®. CONCLUSIONS: The results point to the influence of excipients in Sandimmune® on CyA disposition and more inert nature of PEO-b-PCL micelles in defining CyA biological interactions.

Development of mucoadhesive hydrogels based on polyacrylic acid grafted cellulose nanocrystals for local cisplatin delivery.

To fabricate a mucoadhesive hydrogel with superior properties for local delivery of cisplatin (CDDP) to colorectal cancer, a hardcore bottle-brush polymer (HCBBP) was developed through grafting of poly(acrylic acid) (PAA) on cellulose nanocrystals (CNC) at 6, 9 and 12 CNC:PAA w/w ratios. The developed materials were characterized by acid-base titrations, FT-IR, electron microscopy, muco-rheological behaviour in the presence of mucin, in vitro drug release and anticancer activity against human HCT-116 colorectal cancer cells. The results showed CNC-g-PAA9 to have superior rheological behavior in the presence of mucin compared to CNC and other gels under study indicating beneficial mucoadhesive characteristics. CNC-g-PAA9:CDDP complex showed slow CDDP release causing a significant increase in IC 50 of the drug (> 3-fold) against HCT116 cells. The developed CNC-PAA9 hydrogel showed no intrinsic cytotoxicity on its own. The results point to a great promise for CNC-g-PAA9 as mucoadhesive hydrogels for local platinum delivery in colorectal cancer.

Nano-Delivery of a Novel Inhibitor of Polynucleotide Kinase/Phosphatase (PNKP) for Targeted Sensitization of Colorectal Cancer to Radiation-Induced DNA Damage.

Inhibition of the DNA repair enzyme polynucleotide kinase/phosphatase (PNKP) increases the sensitivity of cancer cells to DNA damage by ionizing radiation (IR). We have developed a novel inhibitor of PNKP, i.e., A83B4C63, as a potential radio-sensitizer for the treatment of solid tumors. Systemic delivery of A83B4C63, however, may sensitize both cancer and normal cells to DNA damaging therapeutics. Preferential delivery of A83B4C63 to solid tumors by nanoparticles (NP) was proposed to reduce potential side effects of this PNKP inhibitor to normal tissue, particularly when combined with DNA damaging therapies. Here, we investigated the radio-sensitizing activity of A83B4C63 encapsulated in NPs (NP/A83) based on methoxy poly(ethylene oxide)-b-poly(α-benzyl carboxylate-ε-caprolactone) (mPEO-b-PBCL) or solubilized with the aid of Cremophor EL: Ethanol (CE/A83) in human HCT116 colorectal cancer (CRC) models. Levels of γ-H2AX were measured and the biodistribution of CE/A83 and NP/A83 administered intravenously was determined in subcutaneous HCT116 CRC xenografts. The radio-sensitization effect of A83B4C63 was measured following fractionated tumor irradiation using an image-guided Small Animal Radiation Research Platform (SARRP), with 24 h pre-administration of CE/A83 and NP/A83 to Luc+/HCT116 bearing mice. Therapeutic effects were analyzed by monitoring tumor growth and functional imaging using Positron Emission Tomography (PET) and [18F]-fluoro-3'-deoxy-3'-L:-fluorothymidine ([18F]FLT) as a radiotracer for cell proliferation. The results showed an increased persistence of DNA damage in cells treated with a combination of CE/A83 or NP/A83 and IR compared to those only exposed to IR. Significantly higher tumor growth delay in mice treated with a combination of IR and NP/A83 than those treated with IR plus CE/A83 was observed. [18F]FLT PET displayed significant functional changes for tumor proliferation for the drug-loaded NP. This observation was attributed to the higher A83B4C63 levels in the tumors for NP/A83-treated mice compared to those treated with CE/A83. Overall, the results demonstrated a potential for A83B4C63-loaded NP as a novel radio-sensitizer for the treatment of CRC.

Development of Self-Associating SN-38-Conjugated Poly(ethylene oxide)-Poly(ester) Micelles for Colorectal Cancer Therapy.

The clinical use of 7-ethyl-10-hydroxy-camptothecin (SN-38), which is the active metabolite of irinotecan, has been hampered because of its practical water-insolubility. In this study, we successfully synthesized two self-associating SN-38-polymer drug conjugates to improve the water-solubility of SN-38, while retaining its anticancer activity. The polymeric micellar SN-38 conjugates were composed of either methoxy-poly(ethylene oxide)-block-poly(α-benzyl carboxylate-ε-caprolactone) conjugated to SN-38 at the PBCL end (mPEO-b-PBCL/SN-38) or mPEO-block-poly(α-carboxyl-ε-caprolactone) attached to SN-38 from the pendent-free carboxyl site (mPEO-b-PCCL/SN-38). The chemical structure of block copolymers was confirmed by 1H NMR. The physicochemical characterizations of their self-assembled structures including size, surface charge, polydispersity, critical micellar concentration, conjugation content and efficiency, morphology, kinetic stability, and in vitro release of SN-38 were compared between the two formulations. In vitro anticancer activities were evaluated by measuring cellular cytotoxicity and caspase activation by MTS and Caspase-Glo 3/7 assays, respectively. The hemolytic activity of both micellar structures against rat red blood cells was also measured. The results showed the formation of SN-38-polymeric micellar conjugates at diameters < 50 nm with a narrow size distribution and sustained release of SN-38 for both structures. The loading content of SN-38 in mPEO-b-PBCL and mPEO-b-PCCL were 11.47 ± 0.10 and 12.03 ± 0.17 (% w/w), respectively. The mPEO-b-PBCL/SN-38, end-capped micelles were kinetically more stable than mPEO-b-PCCL/SN-38. The self-assembled mPEO-b-PBCL/SN-38 and mPEO-b-PCCL/SN-38 micelles resulted in significantly higher cytotoxic effects than irinotecan against human colorectal cancer cell lines HCT116, HT-29, and SW20. The CRC cells were found to be 70-fold to 330-fold more sensitive to micellar SN-38 than irinotecan, on average. Both SN-38-incorporated micelles showed two-fold higher caspase-3/7 activation levels than irinotecan. The mPEO-b-PBCL/SN-38 micelles were not hemolytic, but mPEO-b-PCCL/SN-38 showed some hemolysis. The overall results from this study uphold mPEO-b-PBCL/SN-38 over mPEO-b-PCCL/SN-38 micellar formulation as an effective delivery system of SN-38 that warrants further preclinical investigation.

In Vitro and In Vivo Evaluation of Novel DTX-Loaded Multifunctional Heparin-Based Polymeric Micelles Targeting Folate Receptors and Endosomes.

BACKGROUND: The development of biocompatible tumor-targeting delivery systems for anticancer agents is essential for efficacious cancer chemotherapy. Nanoparticles, as drug delivery cargoes for cancer therapy, are rapidly improving to overcome the limitations of conventional chemotherapeutic agents. Heparin-modified nanoparticles are currently being considered as one of the favorable carriers for the delivery of chemotherapeutics to cancer tissues. OBJECTIVE: This study was aimed at evaluating the in vitro and in vivo antitumor activity of a novel targeted, pH-sensitive, heparin-based polymeric micelle loaded with the poorly water-soluble anticancer drug, docetaxel (DTX). The micelles could overcome the limited water solubility, non-specific distribution, and insufficient drug concentration in tumor tissues. METHODS: DTX-loaded folate targeted micelles were prepared and evaluated for physicochemical properties, drug release, in vitro cellular uptake and cytotoxicity in folate receptor-positive and folate receptor-negative cells. Furthermore, the antitumor activity of DTX-loaded micelles was evaluated in the tumor-bearing mice. Some related patents were also studied in this research. RESULTS: The heparin-based targeted micelles exhibited higher in vitro cellular uptake and cytotoxicity against folate receptor over-expressed cells due to the specific receptor-mediated endocytosis. DTX-loaded micelles displayed greater antitumor activity, higher anti-angiogenesis effects, and lower systemic toxicity compared with free DTX in a tumor-induced mice model as confirmed by tumor growth monitoring, immunohistochemical evaluation, and body weight shift. DTX-loaded targeting micelles demonstrated no considerable toxicity on major organs of tumor-bearing mice compared with free DTX. CONCLUSION: Our results indicated that DTX-loaded multifunctional heparin-based micelles with desirable antitumor activity and low toxicity possess great potential as a targeted drug delivery system in the treatment of cancer.

Poly(ethylene glycol)-poly(ε-caprolactone)-based micelles for solubilization and tumor-targeted delivery of silibinin.

Introduction: Silibinin is a naturally occurring compound with known positive impacts on prevention and treatment of many types of human illnesses in general and cancer in particular. Silibinin is poorly water soluble which results in its insufficient bioavailability and lack of therapeutic efficacy in cancer. Here, we proposed to examine the potential of micelles composed of poly(ethylene glycol) (PEG) as the hydrophilic block and poly(ε-caprolactone) (PCL), poly(α-benzylcarboxylate-ε-caprolactone) (PBCL), or poly(lactide)-(PBCL) (PLA-PBCL) as hydrophobic blocks for enhancing the water solubility of silibinin and its targeted delivery to tumor. Methods: Co-solvent evaporation method was used to incorporate silibinin into PEG-PCL based micelles. Drug release profiles were assessed using dialysis bag method. MTT assay also was used to analyze functional activity of drug delivery in B16 melanoma cells. Results: Silibinin encapsulated micelles were shown to be less than 60 nm in size. Among different structures under study, the one with PEG-PBCL could incorporate silibinin with the highest encapsulation efficiency being 95.5%, on average. PEG-PBCL micelles could solubilize 1 mg silibinin in 1 mL water while the soluble amount of silibinin was found to be 0.092 mg/mL in the absence of polymeric micelles. PEG-PBCL micelles provided the sustained release of silibinin indicated with less than 30% release of silibinin within 24 hours. Silibinin encapsulated in PEG-PBCL micelles resulted in growth inhibitory effect in B16 cancer cells which was significantly higher than what observed with free drug. Conclusion: Our findings showed that PEG-PBCL micellar nanocarriers can be a useful vehicle for solubilization and targeted delivery of silibinin.

ELISA-based detection of Open Reading Frame protein 1 in patients at risk of developing lung cancer.

BACKGROUND: Early detection of lung cancer significantly improves survival outcomes. Thus, lung cancer screening for high-risk individuals using low-dose CT scan (LDCT) is recommended. LDCT has several limitations, and often requires invasive follow up. Previously, we have developed an ELISA for measurement of Open Reading Frame 1 protein (ORF1p) in serum. We assessed whether ORF1p can be used as a risk assessment biomarker for patients at high risk for developing lung cancer. PATIENTS: Patients with risk factors for lung cancer were enrolled in our study with consent under IRB approval. A total of 122 patients were included. The lung cancer cohort consisted of 38 patients with varying stages of cancer undergoing treatment. METHODS: ORF1p quantification was performed using our ELISA assay on serum samples. RESULTS: ORF1p was significantly increased in the serum of patients with identified lung nodules compared to those without nodules (P = 0.0007). ORF1p was also significantly increased in patients who were recommended for follow up (P = 0.0004). When comparing the at-risk cohort to patients with lung cancer, there was not a significant difference in ORF1p levels. CONCLUSION: ORF1p can be used to identify patients at high risk of developing lung cancer and may provide an effective, non-invasive risk assessment marker to complement LDCT screening.

Synthesis and Analysis of 64Cu-Labeled GE11-Modified Polymeric Micellar Nanoparticles for EGFR-Targeted Molecular Imaging in a Colorectal Cancer Model.

Polymeric micellar nanoparticles represent versatile and biocompatible platforms for targeted drug delivery. However, tracking their biodistribution, stability, and clearance profile in vivo is challenging. The goal of this study was to prepare surface-modified micelles with peptide GE11 for targeting the epidermal growth factor receptor (EGFR). In vitro fluorescence studies demonstrated significantly higher internalization of GE11 micelles into EGFR-expressing HCT116 colon cancer cells versus EGFR-negative SW620 cells. Azo coupling chemistry of tyrosine residues in the peptide backbone with aryl diazonium salts was used to label the micelles with radionuclide 64Cu for positron emission tomography (PET) imaging. In vivo analysis of 64Cu-labeled micelles showed prolonged blood circulation and predominant hepatobiliary clearance. The biodistribution profile of EGFR-targeting GE11 micelles was compared with nontargeting HW12 micelles in HCT116 tumor-bearing mice. PET revealed increasing tumor-to-muscle ratios for both micelles over 48 h. Accumulation of GE11-containing micelles in HCT116 tumors was higher compared to HW12-decorated micelles. Our data suggest that the efficacy of image-guided therapies with micellar nanoparticles could be enhanced by active targeting, as demonstrated with cancer biomarker EGFR.

Development of a RP-HPLC method for analysis of docetaxel in tumor-bearing mice plasma and tissues following injection of docetaxel-loaded pH responsive targeting polymeric micelles.

BACKGROUND AND PURPOSE: A simple, rapid, and sensitive reversed-phase high performance liquid chromatography (RP-HPLC) method based on liquid-liquid extraction was developed and validated for determination of docetaxel (DTX) in plasma and homogenate tissues of tumor-bearing mice. EXPERIMENTAL APPROACH: Samples were spiked with celecoxib as the internal standard and separation was achieved on a µ-Bondapak C18 HPLC column. The mobile phase consisted of a mixture of acetonitrile/water (40/60 v/v) at flow rate of 1.2 mL/min and the effluent was monitored at 230 nm. RESULTS: Calibration curves were linear over the concentration range of 0.1-10 µg/mL of DTX in plasma and 0.25-50 µg/mL in tissue homogenates with acceptable precision and accuracy. The mean recoveries of the drug from plasma extraction was 94.6 ± 1.44% while those of tissue homogenates ranged from 73.5 ± 3.2 to 85.3 ± 2.8% depending on the type of tissues examined. DTX was stable in biological samples with no evidence of degradation during 3 freeze-thaw cycles and two months of storage at -70 ± 15 °C. The developed HPLC method was applied to quantify DTX in the mouse plasma and tissues after intravenous administration of 7.5 mg equivalent DTX/kg dose of DTX-loaded folic acid-polyethylene glycol-heparin-tocopherol (FA-PEG-HEP-CA-TOC) micelle formulation to female Balb/c mice. CONCLUSION: A simple, sensitive, rapid, accurate, and prudent RP-HPLC method was developed, validated, and applied for DTX determination in plasma and tissues.

STAT3 inhibitory stattic enhances immunogenic cell death induced by chemotherapy in cancer cells.

BACKGROUND: Induction of immunogenic cell death (ICD) is considered a promising strategy for cancer immunotherapy. Stattic is an inhibitor of STAT3, which is found constitutively active in many cancers and plays a major role in cancer progression. OBJECTIVES: In the present study, we proposed to evaluate whether stattic can enhance the effects of chemotherapy in the induction of ICD in cancer cells harboring hyperactive STAT3. METHODS: The growth inhibitory effects of stattic and chemo agents including doxorubicin (DOX) and oxaliplatin (OXP) were evaluated using MTT assay in B16F10 and CT26 cell lines. Flow cytometry was applied to study cell apoptosis and calreticulin (CRT) surface exposure. The levels of high mobility group box 1 (HGMB1), heat shock protein70 (HSP70) and interleukin-12 (IL-12) were measured using ELISA. RESULTS: Treatment of B16F10 and CT26 cells with stattic in combination with DOX resulted in synergistic antitumor effects with combination index being 0.82 and 0.87, respectively. Interestingly, we found a higher level of ICD markers including CRT expression as well as HMGB1 and HSP70 secretion in the cells received combination therapy of stattic and DOX as compared with monotherapies. Moreover, exposure of dendritic cells (DCs) to conditioned media (CM) from cancer cells treated with stattic and/or DOX resulted in secretion of IL-12, which is an indicator of DCs maturation and induction of Th1 response. OXP and stattic monotherapy induced ICD in CT26 cells and stimulated IL-12 secretion by DCs; however, we did not observe a significant increase in the level of ICD in CT26 cells and IL-12 secretion by DCs when CT26 cells were treated with stattic and OXP combination as compared with monotherapy groups. CONCLUSION: These findings indicate that STAT3 inhibitory stattic can increase ICD induced by DOX. Graphical abstract.